鸡淋巴信号活化分子CD150基因的扩增、表达和氨基酸序列分析

Isolation,Expression and Amino Acid Sequence Analysis of Chicken Signaling Lymphocyte Activation Molecule CD150

CD150蛋白是副黏病毒的重要受体,能够促进病毒与宿主细胞结合并感染。本研究旨在克隆鸡CD150基因后进行原核表达,并对获得的氨基酸序列进行详细分析,为后续基因功能研究奠定基础。从原代鸡胚成纤维细胞(CEF)中扩增得到CD150 CDS序列,将其克隆入pMD19-T载体中进行测序鉴定,将其在pET-28b原核表达载体中表达,并制备多克隆抗体,最后,通过生物信息学软件对CD150进行氨基酸序列分析。结果显示:成功克隆大小为999 bp的鸡CD150基因;鸡CD150蛋白以包涵体形式表达;所制备的多克隆抗体能够特异性结合鸡淋巴细胞表面的CD150蛋白;二级结构中α-螺旋占25.83%,β-折叠占23.42%,无规则卷曲占50.75%;跨膜区分析可知,CD150蛋白包括胞外区、胞内区和跨膜区;三级结构以β-折叠为主,包括V和C2两个结构域,不同物种V结构域中存在7个完全保守的氨基酸位点。本研究成功克隆、表达了鸡CD150蛋白,并制备多克隆抗体,为鸡CD150蛋白生物学功能研究检测及其在鸡组织中的表达和分布提供数据支持。

Chicken signaling lymphocyte activation molecule CD150 is an important receptor of Paramyxovirus,which can promote the virus to bind to host cells and facilitate infection.The purpose of this study was to clone the CD150 gene for prokaryotic expression and conduct amino acid sequence analysis,laying a foundation for future studies on gene function.The CD150 CDS sequences were amplified from primary chicken embryo fibroblasts(CEF)and cloned into pMD19-T vector for construction of vector pET-28b.The amino acid sequence of the expressed CD150 was analyzed by bioinformatics software.The results showed that the open reading frame of CD150 gene was cloned successfully with the length of 999 bp.Chicken CD150 protein was expressed as inclusion body form.The polyclonal antibodies specifically detected the CD150 protein on the chicken lymphocyte surfaces.In the secondary structure,α-helix accounted for 25.83%,β-folding for 23.42%and irregular curl for 50.75%,respectively.Transmembrane analysis showed that CD150 protein included extracellular,intracellular and transmembrane regions.The tertiary structure was dominated byβ-folding,including V and C2 domains.There were 7 completely conserved amino acid sites in V domains of different species.Successful cloning and expression of CD150 protein and preparation of polyclonal antibodies in the present study provided a basis for further study on biological functions and distributions of CD150 protein in chicken tissues.